The long-term storage of biological compounds poses a unique challenge, considering that these compounds are usually fragile and vulnerable. Very few biological compounds are sufficiently stable in liquid environment, in solution or suspension, to allow them to be isolated, purified and stored at unrefrigerated conditions, especially at room temperature or higher temperatures as a solution for anything more than a short period of time.
One way to improve the stability of biopharmaceuticals is by converting them into a dry state [17]. Both commercially and practically, storage of biological compounds in dry form carries with it enormous benefits. Successfully dried reagents, materials and tissues have reduced weight and require reduced space for storage not withstanding their increased shelf life. This is not only of use for the final product, like in final lots of vaccines for use within 3 months to 2 year, but also for stockpiling (1-50 year or more years of storage) seedlots, bulks or final lots. Room temperature storage of dried materials is moreover cost effective when compared to low temperature storage options and the concomitant cost. In addition, several routes of delivery, including pulmonary delivery of powders, dermal delivery by coated or dissolving microneedles, parenteral delivery by powders or dissolvable needles depend on sophisticated ways of formulations. There exist several technologies for producing dried biological compounds, including spray drying, vacuum drying, air-drying, coating, foam-drying. One of the oldest and commonly used technique is freeze-drying, also called lyophilization. For a long period of time freeze-drying was seen as more of an art than a science, which hindered a scientific approach and research.
The most commonly used method for preparing solid biopharmaceuticals is lyophilization. This process consists of a freezing step followed by two drying steps, the primary drying where frozen water is removed by sublimation and the secondary drying where the non-frozen ‘bound’ water is removed. Either freezing or drying stresses can modify the thermodynamic stability of biopharmaceuticals and can induce or facilitate protein unfolding. Unfolding can lead to irreversible denaturation of the biopharmaceutical, but may also reduce the storage stability in the dry state [19]. For a stable lyophilisate, excipients serving as stabilizer and/or bulking agents are used. Different compounds, such as sugars, polymers, amino acids and surfactants, have been shown to improve the stability of biopharmaceuticals during lyophilization and subsequent storage [18, 20]. In literature several mechanisms are described how excipients are believed to protect biopharmaceuticals like proteins and vaccines, during freezing, drying and subsequent storage. Understanding the cryo- and lyoprotection mechanisms of different stabilizers is important in the development of a rational formulation and process design for a stable lyophilized vaccine [21].
During freezing, the physical environment of a biopharmaceutical changes dramatically leading to the development of stresses that impact the integrity of the proteinaceous biopharmaceutical. The most critical stresses to which a biopharmaceutical is exposed during freezing are low temperature, freeze-concentration and the formation of ice [18, 19, 21, 22]. Cold denaturation is the phenomenon whereby biopharmaceuticals lose their compact folded structure as a result of a temperature drop. The currently accepted explanation for cold denaturation is based on a change in the contact free energy between water and non-polar groups at colder temperatures, which would weaken the hydrophobic interaction and thus disrupt biopharmaceutical structure [19, 20, 23]. Due to ice formation, the concentration of all solutes increases dramatically during freezing. All changes related to concentration, such as ionic strength, crystallization of solutes and phase separation, may potentially destabilize a biopharmaceutical [21].
The initial relative composition and pH of the formulation can circumvent detoriation of the biopharmaceutical by freezing stresses. For example, it has been found with many biopharmaceuticals that increasing the biopharmaceutical concentration in the formulation relatively to other excipients before freezing will increase the stability of the biopharmaceutical during freeze-thawing [24]. Similarly, an initial pH that is optimal for the biopharmaceutical in solution will give the highest recovery of intact biopharmaceutical after freeze-thawing [20].
Even after optimization of all these factors, many biopharmaceuticals still denaturize during freeze-thawing, therefore additives are needed to minimize protein/biopharmaceutical denaturation. Different excipients that come from very dissimilar chemical classes are able to give cryoprotection. According to the ‘solute exclusion hypothesis’, cryoprotectants have been shown to preferentially not to be in contact with the surface of biopharmaceuticals in aqueous solutions [24]. The thermodynamic phenomenon of solute exclusion in the presence of various biopharmaceuticals has been determined for various excipients, such as salts, amino acids, methylamines, polyethylene glycols, polyols, surfactants and sugars [19, 21, 24, 25].
The ‘vitrification hypothesis’ is a widely known kinetic mechanism. According to this mechanism, both freeze-concentration and a temperature drop increase viscosity, reduce mobility and slow all dynamic processes. When the system reaches a glassy state, all molecules in the glass become physically (e.g. denaturation, aggregation) and chemically (e.g. oxidation, hydrolysis, deamidation) immobile and the rate constant of biopharmaceutical degradation is reduced [19, 26].
Ice-water interfaces formed during freezing may cause surface denaturation. Addition of surfactants may drop surface tension of the biopharmaceutical solution and thus reduce biopharmaceutical adsorption and aggregation [25, 27].
Polymers could stabilize biopharmaceuticals by raising the glass transition temperature of the formulation and by inhibiting crystallization of small stabilizing additives, like disaccharides [18, 22]. Amino acids may protect biopharmaceuticals as well from freezing denaturation by reducing the rate and extend of buffer salt crystallization and thus suppressing the pH shift [18].
In an aqueous solution biopharmaceuticals are fully hydrated, which means that the biopharmaceutical has a monolayer of water covering and interacting (by hydrogen bonds) with the biopharmaceutical surface [28]. Drying removes part of the hydration shell and this may disrupt the native state of the biopharmaceutical leading to denaturation. In order to prevent denaturation during drying protectants are required. An important stabilization mechanism of such protectants is called the ‘water substitution hypothesis’ [18-20, 29]. Sugars, such as sucrose and trehalose, polyols [20, 30] and amino acids [31] are able to form hydrogen bonds with the dried biopharmaceutical. As such they can act as a water substitute, when the hydration shell is removed. The formation of an amorphous glass, explained above as the ‘vitrification hypothesis’, is also a major protection mechanism.
In addition to water substitution and glass formation, many excipients, especially polymers can stabilize biopharmaceuticals by increasing the glass transition temperature (Tg), which is defined as the transition temperature between the rubbery (liquid-like) and glassy (solid-like) states. Generally, the higher the Tg, the lower the molecular mobility in the glass (e.g. movement of the biopharmaceutical, stabilizing compounds, oxygen and water) and the more stable the biopharmaceutical formulation during drying and subsequent storage [18, 22]. Another mechanism involved in the stabilization of biopharmaceuticals during drying and that is applicable for polysaccharides and other polymers, is the inhibition of crystallization during solute concentration of small excipients that stabilize the biopharmaceutical during drying.
Due to their preference for the bulk environment instead of the biopharmaceutical surface (previously named as ‘solute exclusion’), some excipients are able to act as bulking agent, which means that they provide mechanical support to the final cake, improve product elegance, and prevent product collapse during drying. Mannitol and glycine are frequently used bulking agents, because of their non-toxicity, high solubility, and high eutectic temperature [18, 25, 32]. Most amino acids are potential bulking agents as well as they easily crystallize [33].
If the biopharmaceutical is stable during the drying process, then long-term storage in the dried state is often feasible. Although in general the drying process itself is most detrimental to the biopharmaceutical, dried biopharmaceuticals may still loss their structure or potency during storage if not properly formulated. Aggregation is a major physical instability for biopharmaceuticals during storage. Different chemical degradations, like deamidation, oxidation and hydrolysis, may occur as well during storage, but these alterations may not necessarily affect the activity of biopharmaceutical, depending on the location of the transformed residue(s). Reducing sugars such as glucose and sucrose can react with lysine and arginine residues in biopharmaceuticals to form carbohydrate adduct via the Maillard reaction, a browning reaction, which can lead to a significant loss of activity of the lyophilized biopharmaceutical during storage.
Storage temperature is one of the most important factors affecting the stability of biopharmaceuticals in the solid state. Other factors that affect long-term storage stability are the glass transition temperature (Tg) of the formulation, formulation pH and the residual moisture content after drying. The moisture content of a dried formulation may change significantly during storage due to several factors, including stopper moisture release, crystallization of an amorphous excipients or moisture release from an excipient hydrate. Excipients that are used to stabilize biopharmaceuticals during drying may destabilize biopharmaceuticals in the solid state if their quantities are not appropriately used in the formulation. Also the risk of crystallization of amorphous excipients exist during storage, because the crystalline state is thermodynamically more stable than the glassy state [39]. Many sugars and polyols have the tendency to crystallize, but this is strongly affected by their relative amount in the formulation, storage temperature and relative humidity [18, 40]. In addition, the relative composition of several excipients and the presence of non-crystallizing stabilizers, such as polymers, may inhibit crystallization of excipients.
Stabilization mechanisms for biopharmaceuticals in the dry state during long-term storage are similar to those for lyoprotection, including formation of an amorphous glassy state, water substitution, and hydrogen bonding between excipients and the biopharmaceutical [41]. A combination of these mechanisms is required for maximum biopharmaceutical stabilization in the solid state [18, 42]. The final quality of a lyophilized product is determined by the choice of excipients, including buffering, bulking and stabilizing agents, and the lyophilization process.
Poliomyelitis is a highly infectious disease which mainly affects young children. The disease, caused by any one of three serotypes of poliovirus (type 1, type 2 or type 3) has no specific treatment, but can be prevented through vaccination. Currently, the oral poliomyelitis vaccine (Sabin OPV) is the vaccine of choice to strive for global eradication of poliomyelitis. However, a major concern is the ability of OPV to revert to a form that can cause paralysis, so-called vaccine associated paralytic poliomyelitis (VAPP). Another risk of permanent use of live attenuated poliovirus is the reversion to vaccine-derived polioviruses (VDPV) [1]. In Western countries the use of an inactivated Salk polio vaccine (IPV) is the current preferred way to eliminate the risk of VAPP and circulating VDPV. IPV is thought to be most suitable for continuation of the global eradication program [1-3].
To achieve global polio eradication an (improved) IPV must be efficacious, inexpensive, safe to manufacture, and easy to administer [4]. The feasibility of current IPV in developing countries is limited, because IPV is more expensive than OPV and is administered through injections only [1, 3, 5]. In order to limit the expenses of IPV, WHO and RIVM are developing a non-commercial IPV for technology transfer to developing countries. Because the containment of the wild-type Salk polio virus during production might be an issue, especially in developing countries, the new vaccine will be based on the OPV strain, Sabin (sIPV), for which the production costs is also expected to be less expensive For further reduction in costs, RIVM is developing sIPV formulations that show dose sparing by using an adjuvant and/or other immunization routes [6].
Since alternative delivery methods and improved vaccine formulations have the potential to make vaccine delivery easier and safer [7, 8], currently several alternative vaccine delivery methods are being developed.
It appears that there are differences in heat stability between the various inactivated polio serotypes, with type 1 being the most vulnerable. In the absence of any preservative type 1 deteriorates slowly after storage for two years at 4° C., while type 2 and 3 remain potent for many years. The D-antigen content drops significantly after 20 days at 24° C. and is undetectable after exposure to 32° C. for the same period. In contrast, no significant changes in D-antigenicity were observed for type 2 at either of these temperatures. Type 3 remains stable for 20 days at 24° C., but the D-antigen content drops significantly at 32° C. [9].
All three serotypes of IPV show satisfactory maintenance of potency when incorporated into combined vaccines and stored at 4° C. for periods ranging from one year to over four years, based on observations made on DT-polio vaccine, which is preserved with 2-phenoxy-ethanol and adsorbed to aluminium hydroxide [9, 10]. Longer storage resulted in a decline in antigenicity, especially for type 1 [9]. The IPV as stand-alone vaccine is stable for 4 years at 4° C. and one month at 25° C. [10]. At 37° C. there is a significant loss of potency of type 1 after 1-2 days and of types 2 and 3 after two weeks [11, 12]. Also freezing has a negative effect on the potency of IPV, which is related to loss of the D-antigen structure [12].
After polio eradication a stockpile of polio vaccines is required to anticipate on the potential risk of new polio outbreaks caused by circulating VDPV (even after OPV cessation) [13-15] or bioterrorism attacks. In order to achieve an optimal vaccine stockpile various issues need to be considered. The shelf-life is an important detail, because a delayed expiration time will reduce the stockpile costs [16]. To guarantee the potency of vaccines for many years the shelf-life of vaccines such as IPV needs to be extended. There is thus a need for improved formulations that extend shelf-life of biopharmaceuticals and improved method for producing such formulations, preferably in dry form.